The long term goal is to elicit broadly neutralizing antibodies, but to forward this objective by defining the Env-specific, vaccine-elicited B cell responses in primates primarily directed at the CD4 binding site. Using this information, through iterative design, we will improve responses directed at this and other conserved neutralizing determinants. Project 1, entitled Immunogen Design and Selection, will generate the HIV envelope glycoprotein-based candidate vaccine immunogens to be analyzed in the HIVRAD. The immunogens will be derived by structure-based design, as well as by selection, and responses to them will be analyzed in the other program components. Project 2, B cell Analysis and Ig Genetics, will examine the diversity of the memory B cell repertoire against selected regions of Env elicited by soluble trimers at both short and longer intervals, by newly designed trimeric imijnunogens, as well as long-lived plasma cells in the bone marrow to examine the impact of regimen on V gene usage and somatic hypermutation. Project 3, Memory B Cell Isolation and Antibody Characterization, will isolate B cells using Env-specific FACS and will characterize the isolated Env-specific antibodies for their epitope specificity to aid immunogen redesign and will also analyze the Env-specific memory IgM compartment following Env-trimer inoculation to look for defects in transition to the IgG compartment. Project 4, Bioinformatics and Structure, will use bioinformatics analysis of deep sequencing of B cell transcripts, obtained from the NHP immunogenicity experiments and to characterize antigen-specific antibody populations that arise in response to immunization. The second focus will be to use x-ray crystallography to determine atomic-level structures of high-interest antibodies in complex with gp120. These efforts will be supported by the Administrative Core A, which will institute a management plan to ensure clear communications within the HIVRAD and the NHP Core B which will house the animals, perform immunogenicity experiments with immunogens from Project 1 and provide samples for the analysis of B cell responses for Projects 2, 3 and 4. RELEVANCE: The overall goal of this HIVRAD, and this Overview, is to elicit broadly neutralizing antibodies to the human pathogen HIV-1. The successful elicitation of such antibodies would be a large step forward toward the goal of generating a broadly effective HIV-1 vaccine and would have a substantial impact on improving human public health. ******************************************************************************************************************** Project 1: Immunogen Design and Selection Project Leader (PL): Richard Wyatt, PHD DESCRIPTION (provided by applicant): Project 1, entitled Immunogen Design and Selection, plays the major role of generating the HIV envelope glycoprotein-based candidate vaccine immunogens to be analyzed in this HIVRAD. In Aim 1, we will design novel trimeric immunogens based upon three distinct strategies. Initially, we will utilize recent mapping data performed in our collaborative group on CD4 binding-site directed (CD4bs) neutralizing antibodies (nAbs) isolated from non-human primates (NHPs) immunized with HIV Env trimeric immungens. We compared the NHP CD4bs nAbs to the known, more broadly nAbs isolated from HIV-1-infected human subjects by alanine scanning mutagenesis ofthe HIV-1 gp120. Using the alanine scan information, we generated divergent mAb footprint patterns between the NHP CD4bs Mabs and the structurally defined footprint of the human bNabs. Armed with this information, we will design stabilized trimers that preferentially present neutralizing epitopes to activate VH genes associated with the infection elicited bNabs and to elicit neutralizing antibody responses by vaccination. In the 2nd approach, we will assess the ability of loop-deleted, conformationally stabilized trimers, which more rapidly elicit CD4bs-directed in rabbits, to do so in NHPs. Improved versions will be assessed for their capacity to elicit CD4bs Mabs or to synergize with full-length trimers to elicit CD4bsdirected B cell responses. In Aim 2, we conjugate cysteine stabilized trimers displaying an optimal antigenic profile to liposomses to the determine if they will increase the durability ofthe antibody response to Env as well as better elicit neutralizing antibodies. Finally, in Aim 3 we select novel trimeric immunogens by directed evolution as Env vaccine candidates. Performed as a complementary approach to rational Env design, Env trimers, stabilized by directed evolution and selection criteria established by Dr. Michael Zwick, will be assessed for their ability to elicit B cell responses in NHPs. In addition, HIV-1 Env trimers selected for recognition by NHP germline equivalents of CD4bs bNabs (e.g. VH1-02 or VH4-59) by directed evolution of virus as established in the Zwick laboratory. RELEVANCE: The major objective of Project 1 is to design novel HIV Env immunogens, an undertaking which is essential for the overall goal of the HIVRAD to elicit broadly neutralizing antibodies to the human pathogen HlV-1. The successful elicitation of such antibodies would be a large step forward toward the goal of generating a broadly effective HIV-1 vaccine and would have a substantial impact on improving human public health.